2.1. Heparin and Inhibition of Angiogenesis
Angiogenesis, the induction of growth of new capillary blood vessels, is important in normal processes such as development of the embryo, formation of the corpus luteum and healing of wounds. It is also an important component in pathological processes such as chronic inflammation, certain immune responses, and neoplasia. It is now accepted that angiogenesis is induced by most malignant tumors and that it is necessary for their continued growth and survival. It is also recognized that angiogenesis is a major component of a number of ophthamological pathologies including such as diabetic retinopathy, retrolental fibroplasia and neovascular glaucoma. Additionally, angiogenesis is now recognized as a major component in other non-neoplastic pathological conditions including rheumatoid arthritis, in which abnormal capillary growth can destroy joint cartilage; hemanogiomas, in which abnormal capillary proliferation appears in newborns and can persist for up to 2 years; angiofibromas which develop in the nasopharynx; and psoriasis, in which excessive proliferation and shedding may be dependent on abnormal capillary growth in the dermis [see Folkman and Klagsbrun, Science 235:442 (1987)].
It has been previously discovered that heparin (or heparin fragments) and cortisone will co-act together to inhibit angiogenesis. This is described in U.S. Pat. application Ser. No. 641,305 filed Aug. 16, 1984, the contents of which are incorporated herein by reference. When administered together to mice with certain kinds of tumors, this combination can inhibit the generation of essential capillary vessels that support tumor growth, and can cause the collapse of the blood supply which supports the tumors. A review of the history of this discovery and of related subject matter is contained in the publication "How is Blood Vessel Growth Regulated in Normal and Neoplastic Tissue?" (G H.A. Clowes Memorial Award Lecture), Judah Folkman, Cancer Research, 46:467 (1986) the contents of which are incorporated herein by reference for background.
Cortisone is an anti-inflammatory agent that by itself does not have the ability to inhibit capillary growth. It has been reported in Shubik et al., J. Nat'l Cancer Inst. 57:769 (1976) that 6 .alpha.-methyl prednisolone partially suppressed tumor angiogenesis in hamster cheek pouches under certain conditions, but tumor growth was not stopped. Many other publications have reported continued growth of tumors even in the presence of large amounts of cortisone. It has also been reported [Gross et al., Proc. Nat'l. Acad. Sci. USA 78:176 (1981)] that medroxyprogestrone, dexamethasone and to a lesser extent cortisone, inhibited tumor angiogenesis in rabbit corneas, while estradiol and testosterone were ineffective.
Aside from cortisone, certain other steroids are now known to successfully suppress angiogenesis when administered together with heparin or certain heparin fragments. The effective steroids have been referred to as "hepar-independent" because heparin was (until now) unique in its effect. The findings and the character of desirable angiostatic steroids has been discussed in "A New Class of Steroids Inhibits Angiogenesis in the Presence of Heparin or a Heparin Fragment", R. Crum, S. Szabo and J. Folkman, Science 230:1375 (1985); and in "Angiostatic Steroids", J. Folkman and D.E. Ingber, Annals of Surgery, 206:374 (1987) incorporated herein by reference for background purposes.
Heparin, a mucopolysaccharide, is a constitutent of various tissues, especially liver and lung, and mast cells in several mammalian species. Chemically, it has been described as an .alpha., .beta.glycosidically linked sulfated copolymer of D-glucosamine and D-glucuronic acid. However, although heparin has been used clinically as an anti-coagulant for half a century, both the exact structure of the precise nature by which it acts in blood anti-coagulation have not been discovered. Much of the difficulty in determining the structure of heparin results from its complexity and the fact that it is not a homogeneous, well-defined substance. Heparin is polydisperse with a molecular weight range from about 5,000 to 40,000. Within a given chain, there are also structural variations such as the varying degrees of sulfation, N-acetylation and C-5 epimerization in the uronic acid residue.
A major disadvantage in the use of heparin with a steroid to inhibit angiogenesis results from the fact that heparins manufactured by different processes and different companies revealed quite different antiangiogenic activities despite similar anticoagulant activities. The precise composition of commercial heparin apparently varies depending on its source and method of manufacture. While some heparins may be combined with cortisone to inhibit angiogenesis, other heparins are not effective as such. In fact, some heparins in order to be effective may be required in such high doses that administration may cause problems due to the anticoagulant activity of heparin. A second disadvantage is that while heparins apparently can inhibit the growth of responsive tumors when administered in the proper dose range and proper ratio to steroid, and even, promote regression at somewhat higher doses and ratios; heparins can also cause resumption of rapid tumor growth when administered at even higher dose levels and ratios to steroid. The apparent presence of both positive and negative regulators of angiogenesis in heparin may create problems in properly administering the drug. An additional disadvantage derives from the anticoagulant activity of heparin, restricting its use to low dosage levels or to oral administration in order to avoid bleeding. Finally because heparin cannot penetrate the corneal membrane, it cannot be topically applied to the exterior of the cornea for its desired antiangiogenic activity.